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Biomedical Optics & Medical Imaging

How a tug of war reveals gene transfer secrets

Using optical tweezers to control a single DNA molecule in a nanopore enables better understanding of forces exerted during membrane passage.
27 October 2010, SPIE Newsroom. DOI: 10.1117/2.1201009.003156

The transport of long chain molecules through membranes is one of the most important processes in living cells. Examples include gene transfer between the cell nucleus and cytoplasm as well as between bacteria. Cross-membrane transport processes are governed by a number of interactions, such as electrostatic repulsion and attraction, van der Waals forces or hydrodynamic effects. A deeper understanding can only be obtained by studying membrane transport on molecular level.

In this context, single-molecule techniques are gaining more and more attention due to their ability to quantify inter-molecular forces. Two prominent examples are optical tweezers and the resistive-pulse technique based on solid-state nanopores. The latter is emerging as a tool for rapid and label-free characterization of molecules in aqueous solutions1 using straightforward ionic current measurements through a small orifice. Solid-state nanopores are also promising candidates for biomimetic pores. In addition, optical tweezers have become a workhorse in single-molecule biophysics with sub-piconewton and sub-nanometer resolution.2,3 My colleagues and I have taken the next logical step of combining optical tweezers and solid-state nanopores in a novel technique. We have used this to explore the hydrodynamic interactions on forces during membrane transport.

We first demonstrated the combination by stalling a single DNA molecule in a solid-state nanopore while measuring the ionic current signal.4 This allowed the first direct measurements and control of forces during voltage-driven translocation in sub-10nm nanopores. We found the typical force on a single DNA molecule reached around 24pN at 100mV applied bias voltage.4 It is interesting to note that the force exerted by the optical tweezers enables full control over the DNA translocation velocity. Usually the passage time can only be controlled by reducing the applied voltage, which diminishes the ionic current signal. In addition, optical trapping of the DNA end allows quantitative monitoring of the DNA position with respect to the nanopore with nanometer accuracy. This may enable direct detection of protein binding sites with base-pair resolution. Due to the competition between the electric and mechanical force, this represents a ‘tug of war’ on the molecular scale (see Figure 1).

Figure 1. DNA tug of war. A single DNA molecule can be stalled in a nanopore by tethering it to a colloid in an optical trap. Increasing the nanopore diameter decreases the electrophoretic force on the DNA molecule.

The tug of war enables us to measure the force on the DNA in nanopores with openings ranging from about three to more than 40 DNA molecule diameters (see Figure 1). Our experiments showed that the force depends on the nanopore diameter when holding all other parameters (such as pH and ionic strength) constant. This can be understood by considering that the DNA surface acts as an effective fluid pump when stalled in the nanopore. Due to the no-slip conditions for water molecules at the nanopore walls and DNA surface, the electrophoretic force should depend on the nanopore diameter. The idea is very similar to the pressure drop in a wider or narrower pipe. When the nanopore gets smaller, the fluid flow becomes slower and, thus, counter-intuitively, the detected force on the DNA gets larger (Figure 1). We have demonstrated this experimentally and verified it numerically.5

Aside from the technological challenge, our experiments are relevant for understanding separation of DNA molecules by gel electrophoresis. This technique is of great relevance in biochemistry and molecular biology. Despite the abundance of applications of DNA electrophoresis, the underlying mechanisms remain largely unknown. Meeting this challenge requires detailed studies of the interplay between polymer dynamics, electro-osmosis, the topology of the gel, and many more processes. Combined, optical tweezers and solid-state nanopores are ideally suited to clarify the role of electro-osmosis and hydrodynamic interactions.6 Apart from the relevance for gel electrophoresis, this also has a huge impact on any nanofluidic separation techniques, since our increasing ability to construct nanofluidic systems is just starting to challenge our understanding of the relevant physical processes.7 Since nanopores are ubiquitous in living cells, these findings may shed new light onto the complex cellular processes during membrane transport as well as gel electrophoresis. In fact, trans-membrane potentials of the order of 100mV are common and drive adenosine triphosphate synthesis as well as protein translocation.

In summary, we have developed a novel technique to control single molecules in nanopores. This has allowed us to clarify the role of hydrodynamic interactions on the forces during electrophoretic translocation. It has already been shown that ribonucleic acid (which transfers information from the genome for protein synthesis) as well as DNA-protein complexes can be trapped and studied in nanopores with optical tweezers.8,9 Extending the technique to nanocapillaries10 or biological nanopores, beginning with hybrid samples replacing the native lipid membrane, is an area of active development. In the coming years, we expect optical tweezers to allow us to study biological translocation processes through native cell membranes and possibly even in vivo.11

The author is grateful to have worked with Cees Dekker, Nynke Dekker, Serge Lemay, Stijn van Dorp, Ralph Smeets and many others. Financial support of the Emmy Noether program of the Deutsche Forschungsgemeinschaft is gratefully acknowledged.

Ulrich Keyser
Cavendish Laboratory
University of Cambridge
Cambridge, United Kingdom

Ulrich Keyser pioneered the combination of optical tweezers and solid-state nanopores during his postdoctoral time in the Cees Dekker Lab of the Kavli Institute of Nanoscience at Delft University of Technology. He is head of an Emmy Noether research group and lecturer at the University of Cambridge. His main research focus is single-molecule studies of membrane transport processes.